Journal: PLoS ONE

Article Title: Proliferation and Survival Signaling from Both Jak2-V617F and Lyn Involving GSK3 and mTOR/p70S6K/4EBP1 in PVTL-1 Cell Line Newly Established from Acute Myeloid Leukemia Transformed from Polycythemia Vera

doi: 10.1371/journal.pone.0084746

Figure Lengend Snippet: A–E) PVTL-1 or HEL cells were treated with 1.5 µM JakI-1 or dasatinib at 20 nM (A, C, E) or 15 nM (B, D), as indicated, for 6 h (A, C, E) or 1 h (B, D) and lysed. Total cell lysates were subjected to Western blot analyses using indicated antibodies. Abbreviations used are: Jak2-PY, phospho-Y1007/1008-Jak2; STAT5-PY, phospho-Y694-STAT5; Lyn-PY, phospho-Y396-Lyn; 4EBP1-nonP, non-phospho-T46-4EBP1; 4EBP1-P, phospho-T37/46-4EBP1; Erk-P, phospho-T202/Y204-Erk; mTOR-P, phospho-S2448-mTOR; S6K-P, phospho-T389-p70S6K; S6RP-P, phospho-S240/244-S6RP; eIF4B-P, phospho-S422-eIF4B, GSK3-P, phospho-S21/9-GSK3α/ß. F) PVTL-1 cells were precultured with or without 5 µM SB216763 for 1 h and further cultured with or without 1.5 µM JakI-1 or 20 nM dasatinib, as indicated, for 24 h. Cells were analyzed for cellular DNA content with PI by flow cytometry. Percentages of apoptotic cells with sub-G1 DNA content are indicated. G) PVTL-1 cells were precultured with or without 10 µM SB216763 for 1 h and further cultured with or without 1.5 µM JakI-1 or 20 nM dasatinib, as indicated, for 24 h. Cells were lysed and subjected to Western blot analyses using indicated antibodies. Cl. Casp-3: Cleaved Caspase-3.

Article Snippet: Antibodies against Caspase-3 (CS-9662), cleaved Caspase-3 (CS-9661), phospho-Y705-STAT3 (CS-9131), phospho-Y694-STAT5 (CS-9359), phospho-Y1007/1008-Jak2 (CS-3776), phospho-T202/Y204-Erk (CS-9106), GSK3ß (CS-9315), phospho-S21/9-GSK3α/ß (CS-9331), phospho-S9-GSK3ß (CS-5558), 4EBP1 (CS-9644), non-phospho-T46-4EBP1 (CS-4923), phospho-T37/46-4EBP1 (CS-2855), mTOR (CS-2983), phospho-S2448-mTOR (CS-5536), p70S6K (CS-2708), phospho-T389-p70S6K (CS-9234), phospho-S240/244-S6RP, phospho-S422-eIF4B (CS-3591), Akt (CS-4691), phospho-T308-Akt (CS-9275), and phospho-S473-Akt (CS-9271) were purchased from Cell Signaling Technology (Beverly, MA, USA).

Techniques: Western Blot, Cell Culture, Flow Cytometry

Journal: PLoS ONE

Article Title: Synergistic Effects of Targeted PI3K Signaling Inhibition and Chemotherapy in Liposarcoma

doi: 10.1371/journal.pone.0093996

Figure Lengend Snippet: SW872 and SW982 cells were treated with different concentrations of PI-103 for 24 hours. The effects of PI-103 on PI3K/mTOR pathway protein expression levels were determined by West blot analysis. Note the significant decrease in pAKT and p4EBP1 expression in both of liposarcoma cell lines post PI-103 treatment.

Article Snippet: The human AKT, phosphorylated AKT (pAKT) (threonine [Thr]308), PI3K p110α, phosphorylated 4E binding protein 1 (p4EBP1) (Thr37/46), BcL-XL, Cytochrome c, caspase 3, mTOR, phosphorylated mTOR (pmTOR) antibodies were purchased from Cell Signaling Technologies (Dedham, MA).

Techniques: Expressing

Journal: Comprehensive Psychoneuroendocrinology

Article Title: Differential activation of Gsk-3β in the cortex and the hippocampus induces cognitive and behavioural impairments in middle-aged ovariectomized rat

doi: 10.1016/j.cpnec.2020.100019

Figure Lengend Snippet: Effect of ovx on protein expression in the cortex indicating ( A ) representative Western blot image of mTOR, p -mTOR, Gsk-3β, p -Gsk-3β, β-catenin and β-tubulin. Graphic representation of relative fold change in relation to sham group of ( B ) mTOR ( C ) p -mTOR ( D ) Gsk-3β ( E ) p -Gsk-3β ( F ) β-catenin. n ​= ​7 in each group. Statistical analysis was performed using Mann-Whitney U test for non-parametric data (mTOR expression) and unpaired student t-test for parametric data (all the remaining parameters). ∗P ​< ​0.05 as compared to sham . sham : Group subjected to surgery; ovx : Group subjected to ovariectomy; mTOR: mammalian target of rapamycin; p -mTOR: phosphorylated-mammalian target of rapamycin; Gsk-3β: Glycogen synthase kinase-3 beta; p -Gsk-3β: phosphorylated- Glycogen synthase kinase-3 beta, β-catenin: beta catenin; β-tubulin: beta-tubulin and; A.U. : Arbitrary unit.

Article Snippet: The membrane was blocked in 5% non-fat dried milk (Bio-Rad Laboratories, USA) for 1 h at room temperature, washed thrice in PBST and probed overnight at 4 °C with primary antibodies [Gsk-3β (1:1500,# MA5-15109, Invitrogen, USA), p -Gsk-3β (Ser 9 ) (1:1500,# MA5-14873, Invitrogen, USA), β-catenin (1:4000,# PA5-19469, Invitrogen, USA), mTOR (1:1000,# orb 14604, Biorbyt Ltd., UK), p -mTOR (Ser 2448 ) (1:1000,# SC-293133, Santa Cruz)].

Techniques: Expressing, Western Blot, MANN-WHITNEY

Journal: Comprehensive Psychoneuroendocrinology

Article Title: Differential activation of Gsk-3β in the cortex and the hippocampus induces cognitive and behavioural impairments in middle-aged ovariectomized rat

doi: 10.1016/j.cpnec.2020.100019

Figure Lengend Snippet: Effect of ovx on protein expression in the hippocampus indicating ( A ) representative Western blot image of mTOR, p -mTOR, Gsk-3β, p -Gsk-3β, β-catenin and β-tubulin. Graphic representation of relative fold change in relation to sham group of ( B ) mTOR ( C ) p -mTOR ( D ) Gsk-3β ( E ) p -Gsk-3β ( F ) β-catenin. n ​= ​7 in each group. Statistical analysis was performed using unpaired student t-test. ∗P ​< ​0.05 as compared to sham. sham : Group subjected to surgery; ovx : Group subjected to ovariectomy; mTOR: mammalian target of rapamycin; p -mTOR: phosphorylated-mammalian target of rapamycin; Gsk-3β: Glycogen synthase kinase-3 beta; p -Gsk-3β: phosphorylated- Glycogen synthase kinase-3 beta, β-catenin: beta catenin; β-tubulin: beta-tubulin and; A.U. : Arbitrary unit.

Article Snippet: The membrane was blocked in 5% non-fat dried milk (Bio-Rad Laboratories, USA) for 1 h at room temperature, washed thrice in PBST and probed overnight at 4 °C with primary antibodies [Gsk-3β (1:1500,# MA5-15109, Invitrogen, USA), p -Gsk-3β (Ser 9 ) (1:1500,# MA5-14873, Invitrogen, USA), β-catenin (1:4000,# PA5-19469, Invitrogen, USA), mTOR (1:1000,# orb 14604, Biorbyt Ltd., UK), p -mTOR (Ser 2448 ) (1:1000,# SC-293133, Santa Cruz)].

Techniques: Expressing, Western Blot

Journal: World Journal of Gastroenterology

Article Title: Antagonizing adipose tissue-derived exosome miR-103-hepatocyte phosphatase and tensin homolog pathway alleviates autophagy in non-alcoholic steatohepatitis: A trans-cellular crosstalk

doi: 10.3748/wjg.v29.i29.4528

Figure Lengend Snippet: The capacity of miR-103 in targeting phosphatase and tensin homolog gene and affecting autophagy. A: The luciferase reporter assay results verified the interaction between miR-103 and phosphatase and tensin homolog; B: Western blotting showed the differential expression of the autophagy-related protein; C: Transmission electron microscopy images of autophagosomes (red arrowhead) in the liver; D: The results of immunofluorescence staining showed liver autophagosomes in different groups. a P < 0.01 vs control; b P < 0.01 vs model; c P < 0.01 vs miR-103; d P < 0.05 vs model. PTEN: Phosphatase and tensin homolog; WT: Wild type; MUT: Mutant; DAPI: 4’,6-diamidino-2-phenylindole; mTOR: Mammalian target of rapamycin; NC: Negative control.

Article Snippet: The membranes were blocked and incubated overnight with antibodies against PTEN (9188T, CST), p-AMPK (ab32047, Abcam), p-mammalian target of rapamycin (mTOR) (CSB-PA271384, Cusabio), LC3 (12741T, CST), p62 (ab91526, Abcam), and GAPDH (ab245355, Abcam) at 4 °C.

Techniques: Luciferase, Reporter Assay, Western Blot, Quantitative Proteomics, Transmission Assay, Electron Microscopy, Immunofluorescence, Staining, Control, Mutagenesis, Negative Control

Journal: World Journal of Gastroenterology

Article Title: Antagonizing adipose tissue-derived exosome miR-103-hepatocyte phosphatase and tensin homolog pathway alleviates autophagy in non-alcoholic steatohepatitis: A trans-cellular crosstalk

doi: 10.3748/wjg.v29.i29.4528

Figure Lengend Snippet: The effect of adipose tissue-derived exosomes miR-103 on autophagy in mice. A and B: Western blotting detected the expression of the autophagy-related protein; C: The results of immunofluorescence staining to observe autophagosomes in the liver from different groups; D: Transmission electron microscopy images of autophagosomes (red arrowhead) in the liver from different groups. a P < 0.05 vs control; b P < 0.01 vs control; c P < 0.05 vs exosomes; d P < 0.01 vs exosomes. Exo: Exosomes; PTEN: Phosphatase and tensin homolog; DAPI: 4’,6-diamidino-2-phenylindole; mTOR: Mammalian target of rapamycin; NC: Negative control.

Article Snippet: The membranes were blocked and incubated overnight with antibodies against PTEN (9188T, CST), p-AMPK (ab32047, Abcam), p-mammalian target of rapamycin (mTOR) (CSB-PA271384, Cusabio), LC3 (12741T, CST), p62 (ab91526, Abcam), and GAPDH (ab245355, Abcam) at 4 °C.

Techniques: Derivative Assay, Western Blot, Expressing, Immunofluorescence, Staining, Transmission Assay, Electron Microscopy, Control, Negative Control